MOLECULAR DETECTION OF FIRST-LINE ANTI-TUBERCULOSIS DRUG RESISTANCE GENES IN MYCOBACTERIUM TUBERCULOSIS USING PCR AND AGAROSE GEL ELECTROPHORESIS

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a major global health threat and is becoming more serious with the emergence of drug-resistant strains. In this study, polymerase chain reaction (PCR) and agarose gel electrophoresis were used to investigate the molecular detection of first line anti-tuberculosis drug resistance genes (rpoB, katG, inhA  and embB). A total of 110 newly diagnosed pulmonary TB patients were recruited from selected health facilities in Bayelsa State, Nigeria. Sputum samples were collected and decontaminated using N-acetyl-L-cysteine–sodium hydroxide (NALC–NaOH) and processed for Ziehl–Neelsen microscopy and GeneXpert MTB/RIF assay for initial confirmation. Genomic DNA was isolated using a commercial kit and the target resistance genes were amplified using gene specific primers. PCR products were resolved on a 1% agarose gel and visualised under a blue-light transilluminator. The results indicated that all the target genes were successfully amplified with the expected size of the fragments of 400 bp (rpoB), 200 bp (katG), 500 bp (inhA ) and 300 bp (embB). Multiple isolates of the rpoBgene consistently showed the presence of rifampicin resistance-associated mutations. The katG gene was also frequently amplified, indicating widespread isoniazid resistance via high-level resistance mechanisms. Conversely, the majority of isolates showed no or weak inhA  amplification suggesting a lower prevalence of promoter-mediated isoniazid resistance. Several isolates harboured the embB gene, suggesting the circulation of ethambutol resistance-associated mutations. Resistance-associated genes were more dominant in males and in individuals between 25 and 46 years of age, according to the analysis of gender and age distribution. This study showed that PCR and agarose gel electrophoresis can be used for the rapid detection of genes associated with TB drug resistance. The results indicate the dominance of rpoBand katG mutations in the study population and the importance of molecular diagnostics in improving tuberculosis control strategies and providing suitable therapeutic interventions in Nigeria.